ErbB receptors are tyrosine kinases that regulate colonocyte growth. Increases in EGFR (ErbB1), ErbB2 and several ErbB ligands in colon cancer suggest ErbB derangements contribute to colonic carcinogenesis. The azoxymethane (AOM) rat model of colon cancer recapitulates many features of human colon cancer, including alterations in ErbB signaling. In the prior grant cycle examining Ras activation, we identified AOM tumors with activated wild type Ras and increased expression of ErbB2, a Ras activator. In recent preliminary studies we showed EGFR, and several EGFR effectors were activated in AOM premalignancy. Moreover, Iressa, a specific EGFR antagonist, significantly inhibited (>80%) formation of aberrant crypt foci (ACF), putative precursors of colon cancer. We hypothesize that dys-regulated ErbB signals play important roles in colonic tumor initiation and growth. In the next cycle using the AOM model we will reveal potentially causal changes in ErbB signaling and examine the role of EGFR using genetic and pharmacological approaches. Caco-2 colon cancer cells express functional ErbB receptors and are widely studied for colonocyte growth and differentiation. Using Caco-2 stable transfectants we will elucidate changes in ligand-activated ErbB signaling induced by dominant negative (DN) ErbB or RNA interference (RNAi) strategies. Specific Aim 1 assess the contributions of ErbB signaling and the role of EGFR in AOM tumorigenesis: a) determine changes in ErbB receptors and ligands that occur in premalignancy and tumors; b) directly examine the role of EGFR in colonic carcinogenesis, using Iressa to block EGFR signaling in the rat; and employing mice homozygous for the Waved-2 (wa2) mutation in the EGFR catalytic domain that greatly attenuates kinase activity; and c) quantify carcinogen-induced activation of EGFR effectors, Src, PI3K and Ras and assess the effects of EGFR blockade on their activations. These studies will reveal potentially causal alterations induced by EGFR in these key EGFR effectors. Specific Aim 2 delineate the causal relationships between ErbB signaling and the biological consequences in Caco-2 cells using DN or RNAi strategies: a) Assess the effects of targeted inhibition of ErbB receptors on TGF-alpha or heregulin-induced ErbB signaling and b) elucidate the biological phenotypes, including alterations in growth, apoptosis, tumorigenicity and invasiveness, induced by inhibition of specific ErbB receptors. With these studies, we will further elucidate the roles of ErbB signals in colonic carcinogenesis that can be exploited by selective targeting of receptors (e.g., EGFR blockade by Iressa) or effectors in future strategies to prevent or treat this malignancy.